Method for enhanced protein stabilization and for production of cell lines useful for production of such stabilized proteins

ABSTRACT

A method for increasing production yield of viruses, viral proteins, and other related biological materials through enhanced control and stabilization of protein production via stress proteins and the resultant protein products. The present invention is also directed to methods for selection or engineering of cell lines yielding such enhanced stabilized products. More specifically, example embodiments of the present invention are directed to methods for enhancing production of a viral agent, production of cell lines exhibiting permanent genetic modification, production of permissive eucaryotic cell lines, enhancing functional recombinant product yield, and the products of such methods.

BACKGROUND OF THE INVENTION

[0001] This application claims benefit of copending U.S. provisionalapplication Ser. No. 60/136,676 filed on May 28, 1999 for the sameinventors.

[0002] The present invention relates to the production and stabilizationof functional biological materials, such as proteins, viral agents, orother biological agents or products, including, but not limited to,enzymes, hormones, growth factors, structural proteins, tumor suppressoragents, nucleic acids and nucleic acid probes, vaccines, antigens,antibiotics, lipids, simple and complex carbohydrates, alcohols andother solvents and the products of those methods. Such functionalbiological materials are useful for the manufacture of vaccines anddiagnostic assays or tests, for example.

[0003] The production of viruses, viral antigens, and other viralproducts useful for manufacturing of products such as vaccines anddiagnostic assays, however, is expensive, time consuming and requires ahigh level of technical expertise. Viruses must be propagated in livingeucaryotic cells. Eucaryotic cells that can be infected with aparticular virus are said to be “permissive” for that virus. However,most eucaryotic cells are not permissive for any given virus, and notechniques exist to predict permissiveness. Additionally, cells that arepermissive for virus production may have different levels ofpermissiveness. Some cell lines may produce large amounts of virus whileothers may permit only a low level of viral replication. Therefore, todetermine the optimal cells for virus production, a sometimes large andoften time-consuming empirical study of many cell lines must beperformed. Further complicating matters, the optimal cell line may notbe from the host (e.g. human cells for viruses affecting humans) fromwhich the virus was obtained. Frequently, no suitable cell line existsfor a virus from a particular host.

[0004] Cell lines are preferable to primary cell cultures from a hostbecause of their stability, immortality, known characteristics, andbehavior that has been defined by long experience. The requirements foruse of such cell lines in viral production has led to development of alarge number of exotic cell lines that are used to produce variousviruses. For example, Canine Distemper Virus (CDV), a morbillivirus,grows well in Vero cells (i.e. African Green Monkey kidney cells) but isnot propagated in cells from dogs, while Bovine Leukemia Virus (BLV) isproduced from a cell line derived from bat lung tissue.

[0005] Unfortunately, growth of viruses in cell lines far removed fromthe host and tissue normally infected may cause such viruses to act in afashion that is far removed from their normal behavior.

[0006] Further, the complex pathology for the virus can make themanufacture of viruses extremely difficult. For example, CDV and HumanMeasles Virus (HMV) are closely related viruses. Both viruses actsimilarly when infecting their respective canine and human hosts. Bothproduce encephalitis and long-term sequelae in which the central nervoussystem is damaged or destroyed. The central nervous system disease indogs that occurs many years after initial CDV infection is called olddog encephalitis (ODE). In humans, the corresponding condition issubacute sclerosing panencephalitis (SSPE). The cause of ODE and SSPE isdirectly related to the number of virions is produced and cells affectedduring the encephalitic phase of the disease. This in turn is directlyrelated to permissiveness of the brain cells and the ability of thehost's immune system to rapidly respond to the virus. Dogs or humanswhose brain viral titers reach exceptionally high levels may developthese long-term consequences many years later. In fact, the high viraltiters in some infected patients can result in permanent production of aviral protein in brain cells, even after the infection has cleared. Theimmune system recognizes the viral protein as foreign and continues toattack it even though no viable virus has been produced for many years.Eventually the combat between the host's immune system and theseaberrant cells creates enough damage to destroy the host's cognitivecapacity. Dogs with ODE are usually euthanized. The outcome of SSPE inhumans is a progressive dementia eventually followed by death. Becauseof this complex disease pathophysiology, use of manufactured viruses andviral products has been unsuccessful in stimulating immunity in vivo.

[0007] Since many viruses (such as CDV and HMV) infect brain cells, thepermissiveness of brain cell lines is important in studying thepathophysiology of the resultant disease and in producing viruses fordiagnostics and vaccines that exactly mimic virus characteristics duringnatural infection. However, few neural cell lines exist, and they onlyproduce low levels of CDV or HMV. Therefore, these cell lines produceinadequate levels of the virus for study and are especially unsuitablefor antigen or vaccine production. A method of modifying such cells orcell lines, and the cell lines themselves, is thus required that willsupport replication of viruses and viral products to high levels.

[0008] In addition to the problems associated with producing knownviruses to useful titers, it is extremely difficult to search for anunknown virus because the cell type required for replication of theunknown virus is itself unknown. For example, there are a number ofdiseases of the central nervous system that may well be caused byundiscovered viral agents. The degenerative diseases ofCreutzfield-Jacob Disease (CJD, affecting humans), scrapie (affectingsheep), and bovine spongiform encephalopathy (affecting cattle) all arevery slow neurodegenerative diseases whose etiology remains unknown. Todate, no causative agent for these diseases has been identified nor hasany viral agent been propagated from infected human or animal braintissue. This has lead to the promotion of various unorthodox hypothesesconcerning disease etiology, including suggestions that a specialprotein (called a “prion”, suggested to be an agent capable of causinginfection and reproducing without any genetic material) might serve asthe infectious agent. However, no cell line that produces prions, evento high levels, has been shown to be infectious. Transgenic micemodified to produce prions are not infectious even when they present thediagnostic hallmarks of the disease. Thus, without means foridentification of the infectious agent, no good diagnostic test can beproduced for these diseases, and the production of a vaccine isimpossible.

[0009] Recently, the need for improved ways for development ofdiagnostic tests and potential vaccines has become of pressingimportance. For example, recently cattle in Great Britain were fed mealconsisting of sheep and other animal offal. Bovine spongiformencephalopathy was subsequently recognized for the first time in thesecattle. A number of humans were infected by eating meat from theinfected cattle—constituting a route of infection which had not beenpreviously recognized (now called “new variant CJD”, or nvCJD). Besidesthe tragic consequences to the infected humans, the slaughter of cattlecaused massive economic damage. The finding of nvCJD also producedpolitical repercussions involving the import, export and sale of foodand other animal products that might come from infected cattle.Accordingly, the development of a good diagnostic test is required alongwith the production of an efficacious vaccine. To accomplish these andsimilar goals, permissive cell lines are required that will allow thepropagation of neutrotrophic agents responsible for the etiology of slowdementias like CJD and nvCJD.

[0010] As the average age of a nation's population increases, theincidence of disease states like Alzheimer's also increases. Alzheimer'sdisease is a slowly progressing dementia necessitating difficult,long-term care for the patient. Costs associated with such long-termdebilitating diseases can be devastating. Alzheimer's is not thought tobe infectious. However, certain features of Alzheimer's have causedspeculation that infection with an unconventional viral agent (similarto nvCJD) might cause the disease. It may also be possible that aneurotrophic agent, like HMV or related morbillivirus-like viruses,cause the slow disease process that destroys mentation. If Alzheimer'swere caused by an infectious agents, this would have huge consequencesfor the management, prevention, diagnosis and cure (if possible) of thedisease. Then, for example. prevention of Alzheimer's would likelyrequire the development of a good vaccine. However, as is the case withother dementias produced by unconventional agents (such as ODE andSSPE), no definitive diagnostic test exists. Isolation of a virus frominfected brain tissue would thus be a landmark step in diagnosing andpossibly treating humans with this disease. Hence, cell lines that aremore permissive for viruses like measles may allow the isolation ofneurotrophic agents (like CDV and HMV) not yet discovered.

[0011] Production of proteins from eucaryotic cell lines usingrecombinant DNA technologies for vaccines, diagnostic kits or otherpurposes has the same limitations and problems associated with virusproduction. Recombinant proteins must be produced by eucaryotic celllines to assure proper folding, glycosylation, and other constitutivefactors that are critical to proper function and stability of theprotein or its immunogenicity as a diagnostic reagent or vaccine. Theability to produce these proteins in large quantity while maintainingthe correct conformation is difficult. In many cases, a recombinantprotein may be produced in cell lines at acceptable levels, but due tosome often subtle change in conformation, it is eithernon-immunoreactive as a diagnostic antigen or vaccine or fails toperform its desired function. For example, Canine Parvo Virus (CPV) wasgenetically cloned and produced in a eucaryotic cell line at levelswhich would be commercially feasible. Dogs vaccinated with theserecombinant antigens responded by producing antibodies that could, invitro, neutralize infectious (wild type) parvovirus. Unfortunately, alldogs vaccinated with the recombinant antigen died upon exposure toinfectious CPV. It is likely that some slight conformation changeprevented the recombinant vaccine from protecting the dogs.

[0012] Hence, the production of recombinant proteins is expensive,technically demanding, time consuming and very inefficient. In manycases, recombinant proteins may have useful effects but cannot be usedbecause of the cost involved in their production. For example, numerousanti-tumor peptides are known to have beneficial effects, includingapparent reduction or curing of cancers (for example, angiogenesisblockers). It has been recently claimed that angiostatin and endostatincan cure cancers in mice. However, the cost of producing these peptides(which may run as high as $5-20 million for the quantity of agentnecessary for a single treatment regimen) prevents their use in cancertherapy. Thus, a cost-effective method of producing recombinant peptidesor proteins that maintains their function (enzymic, antigenic, or otherfunctional properties) is desperately needed.

[0013] Many constitutively produced proteins and peptides have closelyassociated “helper proteins” which help induce or maintain proper shape.These helper proteins are often call “chaperones” because they accompanythe proteins through the production process. Some of these chaperoneproteins bind to other proteins to prevent denaturation (loss ofconformation) or other deterioration due to environmental stress. Forexample, the heat shock proteins (such as hsp70 and hsp90) are producedby cells in response to higher than normal levels of heat. Such heatshock proteins bind to other proteins within a cell, stabilizing themand thereby helping to maintain correct conformation and function of thebound protein. These and other, similar proteins that are produced inresponse to other stresses are in general called stress proteins.

[0014] The structure and functional role of such stress proteins appearsto be highly conserved throughout nature, where the various stressproteins appear to play similar chaperone roles in both procaryotic andeucaryotic cells. This has lead to a large number of studies of andproposed uses for such proteins. For example, a number of works in theliterature describe uses for such proteins based on in vitro contact ofvarious biological materials with exogenously-produced stress proteins.These works, which are summarized below, are incorporated herein byreference:

[0015] Neupert et al. (U.S. Pat. No. 5,302,518) suggest that properfolding of proteins may be mediated in vivo by constitutive heat shockproteins, such as GroEL and hsp60 (which occur in E. Coli and ineucaryotic mitochondria, respectively, and which appear to be virtuallyidentical in form and function). Neupert thus describes methods forpost-production modification of the folding of recombinant proteinsbased on in vitro contact of such denatured recombinant proteins withquantities of heat shock proteins that have been isolated from cells.

[0016] Berberian et al. (U.S. Pat. No. 5,348,945) describe methods forenhancement of cell survival under stressful conditions, such methodsconsisting of in vitro or in vivo application (i.e. addition) ofexogenously produced, purified heat shock proteins, such as hsp70, tosuch stressed cells.

[0017] Jacob et al. (U.S. Pat. No. 5,474,892) suggest that certainproteins may be stabilized in aqueous solution via addition ofquantities of certain heat shock proteins (such as hsp90). Jacob et al.thus describes post-production methods for modification of the foldingor other stabilization of various proteins and other biologicalmaterials through in vitro contact of such denatured proteins withquantities of isolated and purified heat shock proteins.

[0018] Srivastava (U.S. Pat. Nos. 5,750,119; U.S. 5,830,464; and U.S.5,837,251) suggests that tumor proliferation in mammals may be inhibitedthrough inoculation of such mammals with antigenic compounds resultingfrom association of certain tumor components with various constitutiveor exogenous stress proteins. Srivastava therefore describes methods forisolation or formulation of such stress protein/tumor complexes usingvarious tumor specimens, and the subsequent inoculation of mammalianpatients with such preparations for the purposes of stimulatinganti-tumor response in such patients.

[0019] Liu et al. (J. Biol. Chem. 13 (1998) 30704) describe studies ofthe role of several heat shock proteins, such as hsp40 and hsp70, inenhancement of protein function. In vitro addition of purl filedexogenously produced heat shock proteins to denatured proteins wasreported to lead to enhanced protein function. A co-chaperone role ofhsp40 with hsp70 was also noted.

[0020] Other pertinent references concerning stress protein function,which are herein incorporated by reference, include:

[0021] McGuire et al. (U.S. Pat. Nos. 5,188,964 and U.S. 5,447,843)describe measurement of the levels of various constitutively producedstress proteins (including the heat shock proteins hsp27, hsp70. andhsp90, and the glucose regulated proteins grp 78 and grp94) present intumor tissues and use of such measurements as a means for predictingprobability of recurrence of such tumors.

[0022] Williams et al. (J. Clin. Invest. 92 (1993) 503) describe meansfor possible protection of cells and tissues from various metabolicstresses, such as ischemia, through transfection with the hsp70 gene.Specifically, constitutively expressed human hsp70 introduced intomurine cells enhanced survival of such modified cells upon applicationof metabolic stress. Addition of such constitutive genes did, however,appear to negatively affect cell proliferation due to the metabolicburden of continual expression, even under unstressed conditions.

[0023] Vasconcelos et al. (J. Gen. Virol. 79 (1998) 1769) describe meansfor induction of enhanced iito expression of constitutive stress proteinprior to infection of permissive Vero cell lines with HMV, leading totransient formation of large plaque phenotype variants of HMV. Cellsinfected 12 hours post stress (where such interval was chosen to allowcells to recover normal physiologic capacity while assuming that inducedheat shock protein levels would be maintained over such interval) werefound to exhibit slightly enhanced HMV production levels (characterizedby up to four-fold increase in viral titer).

[0024] Vasconcelos et al. (J. Gen. Virol. 79 (1998) 2239) describefurther means for transient enhancement of stress protein expressionthrough transfection of permissive cell lines (such as human astrocytomacells) with vectors coding for constitutive hsp72 expression. Clonalcell lines exhibiting uptake of the hsp72 gene were shown to yieldincreased viral titer upon infection with HMV. However, such clonal celllines did not exhibit permanent modification, but rather exhibited amarked tendency to spontaneously regress to the wild-type form. Further,no means for control of expression of the constitutive hsp72 gene isproposed, leading to continuous expression by the modified cell lines.Applicability of the clonal cells produced in this work was limited toHMV—attempts to produce other viral products, for example CDV, wereunsuccessful. Taken in conjunction with the transient nature of themodification, this suggests limited applicability of this approach.

[0025] Yokoyama et. al. (U.S. Pat. No. 5,827,712) describes methods forenhancement of yield in recombinant production of transglutaminase (TG)in E. coli modified for (via transfection) or selected for constitutiveoverexpression of certain chaperone proteins, including DnaJ and DnaK.Such overexpression is used to stabilize and solubilize functionalrecombinant TG. Methods for modification include incubation of stressprotein vector and TG vector with E. coli. Incubation of a combinedstress protein vector/TG vector with E. coli, and incubation of TGvector with E. coli strains exhibiting constitutive overexpression ofstress protein. Notably, such methods are likely to yield transientmodification wherein such stress protein is continuously produced,negatively affecting cell proliferation due to the metabolic burden ofcontinual stress protein expression.

[0026] These examples of prior art clearly establish the role andutility of various stress proteins such as hsp27, hsp40, hsp60, hsp70(including hsp72 and hsp73), hsp90, GroEL, GroES, GrpE, grp 78, grp94,DnaJ and DnaK, as aids to enhanced production and stabilization offunctional biological materials (such as proteins, viral agents, orother biological agents or products, including enzymes. hormones, growthfactors, structural proteins and tumor suppressor agents). However, themethods and concepts taught in these examples, including methods foraddition of exogenously produced stress proteins and for induction oftransient constitutive production of such proteins, are not sufficientlyefficient nor flexible for enhancement of yield in routine production ofvarious biological agents or products, nor for discovery of newbiological agents or products. For example, manufacture and purificationof useful quantities of stress protein for addition to or modificationof product will generally be prohibitively time consuming and expensive.Furthermore, transfection of cells with stress protein vectors codingfor constitutively produced stress proteins will, unless incorporatingsuitable control elements, lead to full-time production of such stressproteins by such cells that will compete with production of desiredbiological products by such cells. Hence, new, more efficient ways forharnessing and controlling the capabilities and use of such stressproteins are required.

SUMMARY OF THE INVENTION

[0027] The present invention is directed to new and more efficientmethods for harnessing and controlling the capabilities and use ofstress proteins in the production and stabilization of functionalbiological materials, such as proteins, viral agents, or otherbiological agents or products, including, but not limited to, enzymes,hormones, growth factors, structural proteins, tumor suppressor agents,nucleic acids and nucleic acid probes, vaccines, antigens, antibiotics,lipids, simple and complex carbohydrates, alcohols and other solventsand the products of those methods. Examples of pertinent stress proteinsof the present invention include, but are not limited to, hsp27, hsp40,hsp60, hsp70 (including hsp72 and hsp73), hsp90, GroEL, GroES, GrpE, grp78, grp94, DnaJ and DnaK. In one embodiment of the present invention,the present invention causes the cells to make such hsp's, as opposed toadding them to the cells.

[0028] Specific preferred embodiments of the present invention include,either through the selective induction of constitutive expression ofsuch stress proteins in or through transient or permanent introductionof such stress protein genes into, procaryotic or eucaryotic cells orcell lines, methods for making various non-permissive cell linespermissive, for increasing yield of various biological products, and fordiscovery and production of various unknown infectious agents.

[0029] For example, the use of such embodiments with such cells or celllines causes dramatic increase in yield of recombinant products inprocaryotic or eucaryotic cell lines. This is surprising, sinceintuitively, one would expect that increasing cellular stress duringproduction would result in decreased productivity. The inventors of thepresent invention have found, however, that by stressing such cells bycertain specific methods or otherwise inducing cellular stress response,thereby leading to controlled expression of stress protein genes,production of functional biological products can be enhanced.

[0030] The present invention is directed to such methods and includesthe five preferred embodiments specifically illustrated herein. Thepresent invention, however, is not limited to the specifics of thesefive embodiments but includes modifications and substitutions within thespirit and scope of the invention, as well as other embodiments whichwill become apparent to those skilled in the art upon reference to thisdescription.

[0031] In the first preferred embodiment, transient stress of aeucaryotic cell line is used to enhance viral titer. Permnissiveeucaryotic cells are transiently stressed for a period sufficient tostimulate production of one or more stress proteins. A desired virusstock is subsequently added following application of this stress so asto infect the stressed eucaryotic cells. Following a period ofpost-infection incubation, the resulting supernatant, containing thedesired virus-induced product, is then harvested.

[0032] In the second preferred embodiment, transient geneticmodification of a eucaryotic cell line AS through episomal insertion ofa stress protein expression vector, followed by selection of one or moresubsets of these modified cell lines that exhibits permanent insertionof the expression vector into host DNA, is used to produce cell linesexhibiting permanent genetic modification. Such cell lines may be usedto enhance production of a desired virus or viral product.

[0033] In the third preferred embodiment, production of a new permissiveeucaryotic cell line is effected through insertion of a stress proteinexpression vector into a non-permissive eucaryotic cell line. The newpermissive cell line is then used to efficiently produce viral agentsthrough inoculation of the cell line with infective or potentiallyinfective material, followed by incubation and harvest of the resultantvirus or viral products thereby produced. Such cell lines arepreferentially used to facilitate replication of difficult to growneural agents and those never cultured before. Hence, such lines may beused both as virus hunters and for production or manufacture of usefulquantities of agent:

[0034] In the fourth preferred embodiment, production of functionalrecombinant product by genetically engineered procaryotic cell lines isenhanced through insertion of one or more stress protein expressionvectors into such cell lines. It is preferred that recombinant cells beselected for use. However, clonal cells may also be selected. It isfurther preferred that such inserted stress protein expression vectorsinclude one or more inducible promoter. Alternatively, a constitutivepromoter can be used. Expression of such stress protein expressionvectors, either by induction or by constitutive expression, in such celllines results in production of the one or more coded stress kiloprotein, wherein such expressed stress protein thereby serves to assistin enhancement of yield of functional recombinant product.

[0035] In the fifth preferred embodiment, production of functionalrecombinant product using genetically engineered eucaryotic cell linesis enhanced through insertion of one or more stress protein expressionvectors into such cell lines. Such insertion may be effected prior to orafter genetic modification of the line for production of the desiredrecombinant product. It is preferred that such stress protein expressionvectors include one or more inducible promoter. Alternatively, aconstitutive promoter can be used.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In describing the preferred embodiments, reference is made to theaccompanying drawings wherein:

[0037]FIG. 1 is an illustration of the use of transient stress ofpermissive eucaryotic cell lines for enhancement of viral titer, inaccordance with the present invention;

[0038]FIG. 2 is an illustration of the permanent genetic modification ofa permissive eucaryotic cell line via transient genetic modification, inaccordance with the present invention;

[0039]FIG. 3 is an illustration of a method for the production of newpermissive eucaryotic cell lines, in accordance with the presentinvention;

[0040]FIG. 4a is an illustration of a method for the enhancement ofrecombinant product yield in genetically engineered procaryotic celllines, in accordance with the present invention; and

[0041]FIG. 4b is an illustration of an alternate method for theenhancement of recombinant product yield in genetically engineeredprocaryotic cell lines, in accordance with the present invention.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0042] The present invention is directed to methods for enhancingproduction of a viral agent, production of cell lines exhibitingpermanent genetic modification, production of permissive eucaryotic celllines, enhancing functional recombinant product yield, and the productsof such methods, as shown in the following embodiments and exampleswhich are not intended to limit but merely illustrate the presentinvention.

[0043] First Preferred Embodiment

[0044] Permissive eucaryotic cells used for production of a desiredvirus or viral product may often be inadequately productive. Theinventors of the present invention, however, have discovered thatproductivity of these cell lines may be transiently enhanced throughapplication of a transient stress. as shown in FIG. 1 and illustrated inthis embodiment.

[0045] In general, in this embodiment, transient stress of a eucaryoticcell line is used to enhance viral titer. More specifically, permissiveeucaryotic cells for the desired virus are selected using conventionalselection methods. Such cells are grown to an approximate confluencyunder standard conditions, and are then transiently stressed for aperiod sufficient to stimulate production of one or more stress protein.Applicable stress factors include thermal stress (such as increased orreduced incubation temperature), chemical stress (such as increased orreduced pH), oxidation level stress (such as increased or reduced levelsof O₂), nutrient modification (such as reduction or enhancement ofessential culture media components), and any other such factors (such asexposure to toxic substances) that can induce transient stress andresultant stress protein expression. A desired virus stock issubsequently added following application of this stress so as to infectthe stressed eucaryotic cells. It is preferred that this addition beperformed at a multiplicity of infection of 0.001-1000 alto 0 virionsper cell, or more preferably at 1-4 virions per cell, and that suchaddition be performed at a period of 0-12 hours post stress, or morepreferably, immediately post stress. The resulting supernatant,containing the desired virus-induced product, is then harvestedfollowing a period of post-infection incubation. It is preferred thatsuch harvest be performed after 1-2 or more days of post-infectionincubation, or more preferably, 5 days post-infection.

EXAMPLE 1 Transient Stress of Eucaryotic Cell Lines for Enhancement ofViral Titer

[0046] Example 1 illustrates an example of the first embodiment. Thepresent invention and the first embodiment, however, are not limited tothe specifics of Example 1.

[0047] Permissive eucaryotic cells (such as Vero cells or otherpermissive eucaryotic cell lines) tor the desired virus (such as CanineDistemper, Mink Distemper, or Human Measles Virus) are selected. Otherviruses to which these cells are permissive include, but are not limitedto, Rabies, Parvo, Marek's agent, HIV, HTLV, HSV, and Corona viruses.Such cells are grown to a confluency of approximately 90% or greaterunder standard conditions, such as at 37° C. with 5% CO₂. The cells arethen transiently stressed for a period of up to several hours, forexample by incubation at 43° C. with no CO₂ (thereby producinghyperthermic alkaline conditions in the culture). This transient stressis ideally applied for a period of approximately 1-5 hours. Enhancedproduction of various stress proteins, such as hsp70, is thereby inducedin such cells. If these cells are subsequently infected with a virus,these increased stress protein levels will serve to stabilize andenhance production of virus-induced proteins by such stressed cells (seefor example Table 3).

[0048] Optimal stress conditions can be determined for a particularcell/virus system based on two related factors: cell viability and viraltiter. While cell viability (the fraction of cells surviving the stressperiod) is in general reduced by application of transient stress, theyield of stress protein (the relative quantity of protein produced, onaverage, by a cell) is in general increased by application of suchstress. Hence, optimal stress conditions exist when cell viabilityexhibits minimal reduction but stress protein yield exhibitssignificantly enhanced expression of protein. Beyond this point,decreased viability can negatively affect overall yield, sincesignificant loss of productive cell numbers can decrease overall proteinproduction capacity of a culture. For example, TABLE 1 comparesviability of Vero cells under various stress conditions. TABLE 2compares protein yield from CDV-infected Vero cells under example stressconditions.

[0049] In Table 1 below, the viability of Vero cell cultures wasestimated using neutral red staining, followed by measurement of theabsorbance (at 570 nm) of resulting samples. Relative viability(non-normalized) is expressed in absorbance units, which corresponds toapproximate level of viable cells following application of transientstress. Test conditions consisted of exposure of cell cultures toindicated temperatures under alkaline conditions (no CO₂) for thespecified duration. TABLE 1 Effects of transient stress severity andduration on cell viability Test Conditions Relative ViabilityTemperature Duration A_(570 nm) 37° C. 1.5 hour 0.25 ± 0.03 37° C. 3hour 0.23 ± 0.03 37° C. 5 hour 0.21 ± 0.03 40° C. 1.5 hour 0.25 ± 0.1040° C. 3 hour 0.22 ± 0.03 40° C. 5 hour 0.19 ± 0.09 43° C. 1.5 hour 0.21± 0.03 43° C. 3 hour 0.18 ± 0.03 43° C. 5 hour 0.13 ± 0.03

[0050] In Table 2 below, the indicated stress conditions were applied toVero cell cultures for a duration of 2 hours, followed by immediate CDVinfection. Infected cell cultures were then incubated under standardconditions (37° C. with 5% CO₂) following this transient stress period.Titer was determined by serial dilution (10× dilution per step) ofharvested supernatant into 96-well plates containing fresh confluentVero cultures. These infected samples were then incubated for 4 days,followed by fixation with neutral buffered formalin. Resultant fixedwells were then stained with fluorescein-conjugated anti-CDV antibodiesand read by cytofluorimetry. Titer results represent number of dilutionsteps resulting in positive viral infection. TABLE 2 Effect of transientstress on virus product yield. Test Conditions log₁₀ (Viral Titer) 37°C. + Alkalinity (pH = 7.6) 3 40° C. + Alkalinity (pH = 7.6) 5 43° C. +Alkalinity (pH = 7.6) 8

[0051] Comparison of the example results shown in TABLES 1 and 2indicate that viral titer in Vero cells is increased significantly at43° C. relative to less stressful culture conditions, and that exposuresof 3 hours or less under such conditions do not significantly impairviability of the test cultures. Hence, for this cell/virus combination,optimal conditions are approximated by stressing cultures at 43° C., 0%CO₂, for two hours immediately prior to viral infection. Further,refinement of these conditions is possible through more detailedevaluation of various stress conditions, not specifically limited tothose conditions or ranges described here. Moreover, for othercell/virus combinations, other parameters will likely be optimal.However, these examples clearly demonstrate the general approach usedfor such optimization. Using such an approach, a skilled practitionercan readily determine the optimal conditions for a specific cell/viruscombination.

[0052] Optimally, the desired virus stock (e.g., CDV) is added to thecells immediately after the stress period, but such cells may beinfected up to 24 or more hours post stress. Virus stock is added at amultiplicity of infection (MOI) of 0.001-1000 virions per cell, or morepreferably at 1-4 virions per cell, the latter levels being moreoptimal. MOI may vary depending on the cell line and virus beingproduced. Following a final incubation period, supernatant is optimallyharvested several days post infection. For Vero cells infected with CDV,such harvest is optimally performed at 5 days post infection, but undersome conditions 1-21 or more days may be required. Optimal conditionsmay be determined by measurement of viral titer as a function of harvesttime. Viral titers thereby produced will be increased 10-100 times ormore, as shown in TABLE 3 (for example, from the initial titer ofapproximately 10⁵ to an enhanced titer of 10⁷⁻⁸). In TABLE 3 below, Verocell cultures were stressed for two hours prior to CDV infection byelevation of temperature to 43° C. under alkaline conditions (no CO₂).Infected cultures were maintained at 37° C. with 5% CO, following thistransient stress period. Control conditions: unstressed culturesmaintained at 37° C. with 5% CO₂. Titer results were obtained in themanner indicated above in TABLE 2. TABLE 3 CDV titer produced bytransient stress Infection Conditions log₁₀ (Viral Titer) Control(unstressed) 4.7 0 hour post stress 8.5 2 hour post stress 7 6 hour poststress 7 12 hour post stress  5

[0053] Second Preferred Embodiment

[0054] Often, permissive eucaryotic cells (such as Vero cells or otherpermissive eucaryotic cell lines) used for production of a desired virusor viral product are inadequately productive. The inventors of thepresent invention, however, have discovered that these cells may begenetically modified so as to enhance yield upon infection with thedesired virus, as shown in FIG. 2 and illustrated in this embodiment.

[0055] In this embodiment, transient genetic modification of aeucaryotic cell line through episomal insertion of a stress proteinexpression vector, followed by selection of one or more subsets of themodified cell lines (resulting from the insertion of the stress proteinexpression vector) that exhibit permanent insertion of the expressionvector into host DNA, is used to produce cell lines exhibiting permanentgenetic modification, as shown in FIG. 2. Such cell lines may be used toenhance production of a desired virus or viral product.

[0056] More specifically, permissive eucaryotic cells for the desiredvirus are selected using conventional selection methods and are grown toan approximate confluency under standard conditions. A DNA transfectionreagent for these eucaryotic cells is prepared by mixing a stressprotein expression vector with a lipid/phospholipid preparation to yielda transfection reagent with a concentration of approximately 0.01-100 μgof vector per 10⁴-10⁶ target cells. It is preferred that thislipid/phospholipid preparation include cationic lipids or phospholipids,and more preferably, that it contain dioleoylphosphatidalethanolamine.This transfection reagent is then delivered to the permissive cellculture, allowing transfection to occur. Following transfection, theculture is incubated in fresh culture media. To increase transfectionyield, the transfection step may subsequently be repeated: The resultantgenetically heterogeneous cell culture is then purified by selection ofthose cell lines exhibiting high-yield expression of the transfectedstress protein and enhanced ability to propagate the desired virus. Fromthis fraction, further purification is performed by selection of thosecell lines exhibiting spontaneous recombination of the expression vectorinto host DNA. Cell lines that exhibit such recombination are thuspermanently genetically modified and are kept for recombinant protein orviral production.

EXAMPLE 2 Permanent Genetic Modification of a Permissive Eucaryotic CellLine via Transient Genetic Modification

[0057] Example 2 illustrates an example of the second embodiment. Thepresent invention and the second embodiment, however, are not limited tothe specifics of Example 2.

[0058] Permissive eucaryotic cells for the desired virus are selectedand grown to a confluency of approximately 90% or greater under standardconditions, such as at 37° C. with 5% CO₂.

[0059] Ads A DNA transfection reagent for these eucaryotic cells isprepared by mixing a stress protein expression vector (such as forexample, a virus, cosmid, plasmid, phage, transposon, or any othertransmissible vector, including DNA or RNA, that is capable of insertioninto eucaryotic cellular DNA and thereby increasing the stress proteinexpression of the eucaryotic cells upon transfection of such cells) witha lipid/phospholipid preparation to yield a transfection reagent with aconcentration of approximately 0.01-100 μg of vector per 10⁴-10⁶ targetcells. As explained above, it is preferred that this lipid/phospholipidpreparation include cationic lipids or phospholipids, and morepreferably, that it contain dioleoylphosphatidalethanolamine. Howeverany other compatible phospholipid may be used in conjunction with one ormore cationic lipid. This transfection reagent is then diluted inserum-free culture medium and incubated on the permissive eucaryoticcells for 10-60 minutes or longer, allowing transfection to occur.Supernatant is then removed from the cell culture, and fresh culturemedium, such as for example fetal bovine serum at a concentration ofapproximately 10% v/v, is added. This culture is then incubated forseveral or more hours, such as overnight. Finally, to further increasetransfection yield, this lipofection procedure may be repeated 24-48hours later. Lipofection is the optimal method for introducing thestress protein expression vector (for example, for expression of hsp70,hsp90, or other stress or chaperone proteins), but other standardtechniques like electroporation may also be used. Genetic modificationusing a viral insertion vector may also be used.

[0060] The transfection process described above produces a geneticallyheterogeneous cell culture in which a fraction of the cells haveundergone transient genetic modification (consisting of episomalinsertion of the stress protein expression vector into the cell withoutsignificant spontaneous recombination of this vector into host cellgenetic material). Such a culture will exhibit a transient geneticmodification (characterized by expression of the episomal vector) butwill generally not include a significant portion of cells exhibitingpermanent modification. Since the episomal vector is not incorporatedinto host cell genetic material, such cultures can spontaneously revertto the uninfected form through expulsion of this episomal geneticmaterial.

[0061] Hence, to obtain a stable culture exhibiting permanentmodification, a subset of these cells must thus be selected andpropagated. For example, transfected cells may exhibit a transient,high-yield expression of stress protein (such as hsp70) due to transientgenetic modification. If the transfected cell cultures are trypsinized,the released cells can be serially diluted to extinction, for example ina 96 well culture plate. Cell cultures that arise in wells near theextinction point are considered to be clonal and are harvested. Thesegenetically modified cell lines are then tested for the ability to growincreased levels of virus (for example, as described in Example 1) andmeasured for enhanced expression of stress protein (for example usingstandard enzyme-linked immunosorbant assay (ELISA) for expression levelsof the particular stress protein, such as hsp70). Cell lines passingthese tests are then probed (using standard assays such as gelelectrophoresis followed by Southern blot assay of the separated nucleicacids) for insertion of the expression vector into host DNA viaspontaneous recombinational events. Note that such insertion occursspontaneously at a rate of approximately 1 recombinational event in1×10³ cell divisions. However, recombinational efficiency may be muchlower depending on specific vector and cell lines used and may thereforenecessitate additional selection steps. Recombinant cell lines thatexhibit a stable insertion into the host cell's genetic material, andare thus permanently genetically modified, are kept for recombinantprotein or viral production lines. Such cell lines will exhibitdesirable improvements in yield of a desired virus or viral product as aresult of the incorporation of the stress protein gene and commensurateenhancement of stress protein production.

[0062] Third Preferred Embodiment

[0063] Often, permissive eucaryotic cells necessary for production of adesired agent, such as a virus or viral product, are non-existent orinadequately productive. For example, culturing of agents that affectthe central nervous system can be very difficult since few cells arepermissive to such agents. Specifically, agents such as theCreutzfeld-Jacob agent, scrapie, Kuru, Rabies, and Bovine SpongiformEncephalopy (nvCJD) are difficult or impossible to culture in vitrobecause of a lack of adequate permissive cell lines. Hence, there is aneed for new process for production of permissive cell lines for suchagents.

[0064] In this embodiment, non-permissive eucaryotic cells aregenetically modified so as to be made permissive, and production of anew permissive eucaryotic cell line is effected through insertion of astress protein expression vector into a non-permissive eucaryotic cellline. The new permissive cell line is then used to efficiently produceviral agents through inoculation of the cell line with infective orpotentially infective material, followed by incubation and harvest ofthe resultant virus or viral products thereby produced. Such cell linesare preferentially used to facilitate replication of difficult to growneural agents and those never cultured before. Hence, such lines may beused both as virus hunters and for production or the manufacture ofuseful quantities of agent.

EXAMPLE 3 Production of New Permissive Cell Lines

[0065] Example 3 illustrates an example of the third embodiment. Thepresent invention and the third embodiment, however, are not limited tothe specifics of Example 3.

[0066] In this example, non-permissive eucaryotic cells, such as insectcells, are genetically modified so as to be made permissive for thedesired virus, as shown in FIG. 3. Neural cell lines, such asneuroblastoma, astrocytoma, or retinoblastoma cell lines, can betransfected with a stress protein expression vector, as described inExample 2. This yields a new cell line capable of efficiently producingviral agents. It is preferred in this process that recombinant celllines be selected for subsequent use. Alternatively, clonal cell linesmay also be used. Infected or potentially infected material, such asanimal offal, containing a known or suspected infectious agent, is thenused to inoculate these genetically modified cell lines. Such cell lineswill facilitate replication of difficult to grow neural agents likerabies and those never cultured before (for example, nvCJD), and may beused both as a virus hunter (used to produce characterizable quantitiesof a suspect infectious agent) and for manufacture of large quantitiesof agent (for example, in the production of a new vaccine). Examples ofnew permissive cell lines produced from non-permissive cell lines areshown in TABLE 4.

[0067] In Table 4 below, there is shown the results of a comparison ofhuman measles virus (HMV) titer raised in native (untreated),non-permissive cell lines (Line A, neuroblastoma; Line B,retinoblastoma) and in the same cell lines following treatment to inserthsp70 stress protein expression vector. Increased titer followingtreatment is indicative of markedly enhanced permissiveness of thesecells to HMV. Enhancement of titer in multiple cell lines is indicativeof the general applicability of this method to various candidate celllines and infectious agents. TABLE 4 Effects of inserted stress proteinexpression vector on viral titer Cell Line Treated log₁₀ (Viral Titer) ANo 1.10 A Yes 6.70 B No 1.60 B Yes 6.30

[0068] Fourth Preferred Embodiment

[0069] Procaryotic cell lines are frequently used for production ofproteins or other biological materials via recombinant methods, i.e.genetic material coding for the production of a desired product isintroduced into the cell line, resulting in the production of thedesired product by the procaryotic cells. For example, human insulin maybe produced using bacteria that have been genetically engineered throughthe introduction of the necessary human genes coding for production ofthe desired insulin proteins. Unfortunately, such recombinant productsoften fail to exhibit the same biological function as the constitutivelyproduced materials (i.e., materials produced via natural means). Thisfrequently is the result of incomplete or improper folding (or changesin other similar factors affecting conformation) during the recombinantproduction process. Such chances in folding or conformation often resultin a denatured protein, negatively affecting proper function of theproduct. Hence, a process for enhancing yield of functional recombinantproduct is needed.

[0070] In this embodiment, production of functional recombinant productby genetically engineered procaryotic cell lines is enhanced throughinsertion of one or more stress protein expression vectors into suchcell lines. It is preferred that recombinant cells be selected forsubsequent use. Alternatively, clonal cells may also be selected. It isfurther preferred that such inserted stress protein expression vectorsinclude one or more inducible promoter. Alternatively, a constitutivepromoter can be used. Expression of such stress protein expressionvectors, either by induction or by constitutive means, in such celllines results in production of the one or more coded stress protein,wherein such expressed stress protein thereby serves to assist inenhancement of yield of functional recombinant product. Such enhancementis optimally achieved through insertion and subsequent induction ofsuitable stress protein expression vectors, such as hsp70 or hsp90, butany other stress protein expression vector is applicable. Such insertionis optimally achieved through transfection. However, electroporation orother similar techniques, along with cell line infection via exposure toone or more viral insertion vectors, may be used. Such enhancementmethods may be used to enhance yield of functional product from apreviously modified cell line, such as, for example one that has beenmodified to produce human insulin. Alternatively, a procaryotic cellalready exhibiting a stable insertion that results in or promotesproduction of one or more stress proteins may be modified to produceviruses or other recombinant products (such as proteins, enzymes,insulin, or other desired biological products).

EXAMPLE 4 Enhancement of Recombinant Product Yield in Procaryotic CellLines

[0071] Example 4 illustrates an example of the fourth embodiment. Thepresent invention and the fourth embodiment, however, are not limited tothe specifics of Example 4.

[0072] As illustrated in this embodiment and examples, the inventors ofthe present invention have discovered an approach for enhancingfunctional recombinant yield that includes induction of one or morestress proteins in the procaryotic cell line, wherein such inducedstress proteins serve to assist proper folding or other conformationalchanges of the recombinant product, thereby resulting in enhanced yieldof functional recombinant product. This induction can be optimallyachieved by insertion of a suitable stress protein expression vector(for example hsp70 or hsp90) into the recombinant procaryotic cell line,such as genetically engineered Escherichia coli. This insertion isoptimally achieved using transfection. Alternatively, electroporation orother similar techniques may be used. This general process isillustrated in FIGS. 4a and 4 b. Note that various viral insertionvectors may also be used to insert desired genetic elements coding foror promoting stress protein production. Insertion of the stress proteinexpression vector can result in recombinant insertion of the stressprotein expression vector into the host genetic material, as illustratedin FIG. 4a, or episomal insertion, as illustrated in FIG. 4b. Theresultant transfected cells are then screened using standard proceduresto select those exhibiting high levels of stress protein expression.Using methods comparable to those used with eucaryotic cell lines, suchas those described in Example 2, recombinant cell lines are selectedthat have a stable insertion into the host genetic material, as shown inFIG. 4a. Alternately, clones exhibiting episomal insertion may be oused, as shown in FIG. 4b. The stress protein expression vector may inthis manner be inserted into a previously modified cell line, such as,for example one that has been modified to produce human insulin.Alternatively, a procaryotic cell already exhibiting a stable insertionthat results in or promotes production of one or more stress proteinsmay be modified to produce viruses or other recombinant products (suchas proteins, enzymes, insulin, or other desired biological products).

[0073] It is preferable in the examples illustrated in FIGS. 4a and 4 bthat the stress protein expression vector include one or more induciblepromoter. Alternatively, a constitutive promoter can be used. Induciblepromoters are more desirable since they allow cell lines to bepropagated to high levels without exhibiting significant expression ofthe inserted stress protein expression vector (such expression wouldtend to compete with expression of the desired recombinant product).Once such cell lines have reached the desired level of recombinantproduct production, induction of the inducible stress protein expressionvector can be used to redirect production of the cell lines to stressprotein, thereby assuring optimum efficiency in recombinant productproduction levels and yield of functional product. Examples of induciblepromoters include, but are not limited to, β-galactosidase, retroviralsteroid-sensitive, and heavy metal inducible promoters. If, in contrast,a constitutive promoter is used, cell lines may tend to devote asignificant portion of production capacity on continuous or nearlycontinuous production of stress protein. Such production will yield ahigh proportion of functional product but will tend to compete withoptimum cell line propagation and total production level of therecombinant product.

[0074] Fifth Preferred Embodiment

[0075] Eucaryotic cell lines may be used for production of desiredproteins or other biological materials or products via recombinantmethods, i.e. genetic material coding for production of a desiredproduct is introduced into the cell line, resulting in production of thedesired product by the eucaryotic cells. For example, human angiogenesisblocking peptides can be produced using eucaryotic cell lines that havebeen genetically engineered through the introduction of the necessaryhuman genes coding for production of the desired products.Unfortunately, such recombinant methods often fail to yield the desiredproducts with acceptable yield or in acceptable quantity. Further, suchproducts may fail to exhibit the same biological function as theconstitutively produced materials. Hence, a method for enhancingproduction level or yield of functional recombinant product is needed.

[0076] In this embodiment, production of functional recombinant productusing genetically engineered eucaryotic cell lines is enhanced throughinsertion of one or more stress protein expression vectors into suchcell lines. Such insertion may be effected prior to or after geneticmodification of the line for production of the desired recombinantproduct. It is preferred that such stress protein expression vectorsinclude one or more inducible promoter. Alternatively. a constitutivepromoter can be used.

EXAMPLE 5 Enhancement of Recombinant Product Yield in Eucaryotic CellLines

[0077] Example 5 illustrates an example of the fifth embodiment. Thepresent invention and the fifth embodiment, however, are not limited tothe specifics of Example 5.

[0078] As illustrated in this embodiment and example, the inventors ofthe present invention have discovered an approach for enhancing yield offunctional recombinant product that includes induction of one or morestress proteins in the eucaryotic cell line, wherein such induced stressproteins serve to assist proper folding or other conformational changesof the recombinant product, thereby resulting in enhanced yield offunctional recombinant product. For example, an expression vector forproduction of a desired biological product (such as an angiogenesisblocking or enhancing peptide sequence) may be inserted into aeucaryotic cell line (such as a mammalian, insect or other cell line,and more preferably S-9 insect cell lines) before or after geneticmodification of the line with a stress protein expression vector codingfor production of a stress protein. Such modification with a stressprotein expression vector (such as hsp70 or hsp90) is effected usingmethods described in Example 2. This stress protein expression vectormay preferably include one or more inducible promoter. Alternatively, aconstitutive promoter can be used. Stress protein thereby induced (as aresult of such modification) facilitates enhanced production andstability of the desired recombinant product. Optimally, this procedureis used for the production of one or more peptides. but it may also beused to internally stabilize proteins required for the synthesis andassembly of a non-peptide product or other biological material, such asdiagnostic nucleic acid probes, vaccines. antigens, enzymes, hormones,growth factors, structural proteins, tumor suppressor agents,antibiotics, lipids, nucleic acids, simple and complex carbohydrates,alcohols and other solvents.

[0079] This description has been offered for illustrative purposes onlyand is not intended to limit the invention of this application.

[0080] What is claimed as new and desired to be protected by LettersPatent is set forth in the appended claims.

We claim:
 1. A method for-enhanced production of a viral agent, saidmethod comprising the steps of: selecting and propagating permissiveeucaryotic cells; transiently stressing said cells for a period of timeto produce stressed eucaryotic cells exhibiting increased production ofat least one stress protein; introducing a virus stock to infect saidstressed eucaryotic cells; incubating said infected stressed eucaryoticcells; and harvesting the resultant viral agent produced by saidinfected stressed eucaryotic cells.
 2. The method of claim 1 whereinsaid step of transiently stressing is induced by stress selected fromthe group consisting of thermal stress, chemical stress, oxidation levelstress, nutrient modification, and toxicity stress.
 3. The method ofclaim 1 wherein said step of transisiently stressing said cells isperformed by stressing said cells at 43° C. with no CO₂.
 4. The methodof claim 1 wherein said step of transiently stressing said cells isperformed for a period between approximately 1 to 5 hours.
 5. The methodof claim 1 wherein said step of transisiently stressing said cells isperformed for Vero cells for a period between approximately 1 to 3 hoursat approximately 43° C. with approximately 0% CO₂.
 6. The method ofclaim 1 wherein said stress proteins are selected from the groupconsisting of hsp 27, hsp 40, hsp 60, hsp 70, hsp 72, hsp 73, hsp 90,GroEL, GroES, GrpE, grp 78, grp 94, DnaJ and Dnak.
 7. The method ofclaim 1 wherein said permissive eucaryotic cells are Vero cells.
 8. Themethod of claim 1 wherein said virus stock is introduced at amultiplicity of infection of between 0.001 to 1000 virons per cell. 9.The method of claim 8 wherein said virus stock is introduced at amultiplicity of infection of between 1 to 4 virons per cell.
 10. Themethod of claim 1 wherein said step of introducing said virus stock isperformed for a period of up to approximately 24 hours after said stepof transisiently stressing said cells.
 11. The method of claim 10wherein said step of introducing said virus stock is performedimmediately after transiently stressing said cells.
 12. The method ofclaim 1 wherein the resultant viral agent is harvested approximately1-21 days after infection.
 13. The method of claim 1 wherein theresultant viral agent is harvested approximately 5 days after infection.14. The method of claim 1 wherein optimal conditions for harvesting theresultant viral agent are determined by measurement of viral titer as afunction of harvest time.
 15. The method of claim 1 wherein said viralagents are selected from the group consisting of Canine Distemper, MinkDistemper, Human Measles Virus, Rabies, Parvo, Marek's agent, HIV, HTLV,HSV, Corona virus, and Bovine Leukemia Virus.
 16. A method forproduction of cell lines exhibiting permanent genetic modification, saidmethod comprising the steps of; selecting and propagating permissiveeucaryotic cells; introducing a transfection reagent to said permissiveeucaryotic cells to produce transfected eucaryotic cells; incubatingsaid transfected eucaryotic cells; and selecting from said transfectedeucaryotic cells at least one first cell line exhibiting high yield of aprotein expressed as a result of transfection by said transfectionreagent.
 17. The method of claim 16 wherein at least one second cellline is produced from said at least one first cell line by selecting aportion of said first cell line exhibiting spontaneous recombination ofthe stress protein expression vector into host DNA.
 18. The method ofclaim 16 wherein said transfection reagent is a DNA transfectionreagent.
 19. The method of claim 16 wherein said transfection reagent isformed of a stress protein expression vector and at least one lipid orphospholipid.
 20. The method of claim 16 wherein said transfectionreagent has a concentration of approximately 0.01-100 μg of vector per10⁴-10⁶ target cells.
 21. The method of claim 16 wherein said stressprotein expression vector is selected from the group consisting of avirus, cosmid, plasmid, phage, transposon, and other nucleic acid whichis capable of insertion into eucaryotic cellular DNA.
 22. The method ofclaim 19 wherein said lipid or phospholipid includes a lipid selectedfrom the group consisting of cationic lipids and phospholipids.
 23. Themethod of claim 19 wherein said lipid or phospholipid includesdioleoylphosphatidalethanolamine.
 24. The method of claim 16 whereinsaid transfected eucaryotic cells are incubated for a period ofapproximately 10 to 60 minutes.
 25. The method of claim 16 furthercomprising the steps of removing supernatant from said transfectedeucaryotic cells and adding a fresh culture medium and furtherincubating said transfected eucaryotic cells.
 26. The method of claim 25wherein said fresh culture medium is a fetal bovine serum in aconcentration of approximately 10% v/v.
 27. The method of claim 25wherein said further incubating is done for several hours.
 28. Themethod of claim 16 wherein said step of introducing a transfectionreagent is repeated.
 29. The method of claim 19 wherein said stressprotein expression vector is selected from the group consisting of hsp70, hsp 72, hsp 73, hsp 90, GroEL, GroES, GrpE, grp 78, grp 94, DnaJ andDnak.
 30. A method for the production of permissive eucaryotic celllines, said method comprising the steps of: inserting a stress proteinexpression vector into a non-permissive eucaryotic cell line to form atleast one permissive eucaryotic cell line; and selecting at least onefirst cell line from said permissive eucaryotic cell line.
 31. Themethod of claim 30 wherein said at least one selected first cell line isused for production of a viral agent, and said method further comprisingthe steps of: introducing an infective material to said selected cellline to produce at least one infected permissive eucaryotic cell line;incubating said at least one infected cell line; and harvesting at leastone resultant viral agent from said infected cell line.
 32. The methodof claim 31 wherein said production is for discovery of at least oneunknown viral agent.
 33. The method of claim 30 wherein saidnon-permissive eucaryotic cell line is selected from the groupconsisting of neuroblastoma, astrocytoma and retinoblastoma cell lines.34. The method of claim 30 wherein said step of inserting is bytransfection.
 35. The method of claim 30 wherein said stress proteinexpression vector is selected from the group consisting of hsp 70, hsp72, hsp 73, hsp 90, GroEL, GroES, GrpE, grp 78, grp 94, DnaJ and Dnak.36. The method of claim 30 wherein said at least one permissiveeucaryotic cell line is recombinant for said stress protein expressionvector.
 37. The method of claim 30 wherein said at least one permissiveeucaryotic cell line is clonal for said stress protein expressionvector.
 38. The method of claim 31 wherein said infective material is ananimal offal containing an infectious agent.
 39. A method for enhancingfunctional recombinant product yield, wherein genetic material has beenpreviously introduced into a procaryotic cell line to form saidrecombinant product, said method comprising the steps of: inserting atleast one stress protein expression vector into said procaryotic cellline so as to form a modified cell line; and selecting from saidmodified cell line at least one first cell line exhibiting enhancedyield of the desired functional recombinant product.
 40. The method ofclaim 39 wherein at least one second cell line is produced from saidfirst cell line by selecting a portion of said first cell lineexhibiting spontaneous recombination of the stress protein expressionvector into host DNA.
 41. The method of claim 39 wherein said at leastone stress protein expression vector includes at least one induciblepromoter.
 42. The method of claim 41 wherein said inducible promoter isselected from the group consisting of β-galactosidase, retroviralsteroid-sensitive promoters and heavy metal inducible promoters.
 43. Themethod of claim 39 wherein said at least one stress protein expressionvector includes a constitutive promoter.
 44. The method of claim 39 whensaid step of inserting is accomplished by a process selected from thegroup consisting of transfection, electroporation, and viral insertionvector.
 45. The method of claim 39 wherein said stress proteinexpression vector is selected from the group consisting of hsp 70, hsp72, hsp 73, hsp 90, GroEL, GroES, GrpE, grp 78, grp 94, DnaJ and Dnak.46. The method of claim 39 wherein said procaryotic cell line isEscherichia coli.
 47. The method of claim 39 wherein at least one secondcell line is produced from said first cell line by selecting a portionof said first cell line exhibiting episomal insertion of the stressexpression vector into the host.
 48. A method for enhancing functionalrecombinant product yield, wherein genetic material coding for suchproduct is introduced into a procaryotic cell line exhibiting enhancedstress protein expression.
 49. The method of claim 48 wherein saidexpression is inducible.
 50. The method of claim 48 wherein saidprocaryotic cell line includes an expression vector that includes atleast one inducible promoter.
 51. The method of claim 48 wherein saidexpression is constitutive.
 52. The method of claim 48 wherein saidprocaryotic cell line includes an expression vector that includes atleast one constitutive promoter.
 53. A method for enhancing functionalrecombinant product yield, said method comprising the steps of:inserting at least one stress protein expression vector into aneucaryotic cell line to make a modified cell line. selecting from saidmodified cell line at least one first cell line exhibiting enhancedyield of the desired functional recombinant product, wherein geneticmaterial is added to said eucaryotic cell line to form said recombinantproduct.
 54. The method of claim 53 wherein said step of inserting atleast one stress protein expression vector into an eucaryotic cell lineoccurs after said genetic material has been added to said eucaryoticcell line.
 55. The method of claim 53 wherein said step of inserting atleast one stress protein expression vector into an eucaryotic cell lineoccurs before said genetic material has been added to said eucaryoticcell line.
 56. The method of claim 53 wherein at least one second cellline is produced from said first cell line by selecting a portion ofsaid first cell line exhibiting spontaneous recombination of the stressprotein expression vector into host DNA.
 57. The method of claim 53wherein said stress protein expression vector includes at least oneinducible promoter.
 58. The method of claim 53 wherein said stressprotein expression vector includes a constitutive promoter.
 59. Themethod of claim 53 wherein said eucaryotic cell line is selected fromthe group consisting of mammalian and insect cell lines.
 60. The methodof claim 53 wherein said stress expression vector is selected from thegroup consisting of hsp 70, hsp 72, hsp 73, hsp 90, GroEL, GroES, GrpE,grp 78, grp 94, DnaJ and Dnak.
 61. The method of claim 53 wherein saidmethod is used to produce functional recombinant yields selected fromthe group consisting of peptides, proteins, diagnostic nucleic acidprobes, vaccines, antigens, enzymes, hormones, growth factors,structural proteins, tumor suppressor agents, antibiotics, lipids,nucleic acids, simple carbohydrates, complex carbohydrates, alcohols andsolvents.
 62. A viral agent produced by the steps of: selecting andpropagating permissive eucaryotic cells; transiently stressing saidcells for a period of time to produce stressed eucaryotic cellsexhibiting increased production of at least one stress protein;introducing a virus stock to infect said stressed eucaryotic cells;incubating said infected stressed eucaryotic cells; and harvesting theresultant viral agent produced by said infected stressed eucaryoticcells.
 63. A cell line selected for exhibition of permanent geneticmodification comprising: a permissive eucaryotic cell line exhibiting ahigh yield of a transfected stress protein as a result of transfectionwith one or more vectors for expression of such protein.
 64. A cell lineexhibiting permanent genetic modification produced by the steps of:selecting and propagating permissive eucaryotic cells; introducing atransfection reagent to said permissive eucaryotic cells to producetransfected eucaryotic cells; incubating said transfected eucaryoticcells; and selecting from said transfected eucaryotic cells at least onefirst cell line exhibiting high yield of the stress protein expressed asa result of said transfection.
 65. A permissive eucaryotic cell linecomprising: a non-permissive eucaryotic cell line into which a stressprotein expression vector has been inserted.
 66. A permissive eucaryoticcell line produced by the steps of: inserting a stress proteinexpression vector into a non-permissive eucaryotic cell line to form atleast one permissive eucaryotic cell line; and selecting said permissiveeucaryotic cell line.
 67. A functional recombinant product produced bythe steps of: introducing a genetic material into a procaryotic cellline coding for expression of said recombinant product; inserting atleast one stress protein expression vector into said procaryotic cellline so as to form a modified cell line; selecting from said modifiedcell line at least one first cell line exhibiting enhanced yield of thedesired functional recombinant product; propagating said first cellline; and collecting functional recombinant product produced by saidpropagated first cell line.
 68. A functional recombinant productproduced by the steps of: inserting at least one stress proteinexpression vector into an eucaryotic cell line to make a modified cellline. selecting from said modified cell line at least one first cellline exhibiting enhanced yield of the desired functional recombinantproduct; propagating said first cell line; and collecting functionalrecombinant product produced by said propagated first cell line.